Virus-like particles useful as a vector for delivering nucleic acid

ABSTRACT

The present invention relates to the use of virus-like particles (VLP&#39;s) of papillomavirus for preparing vector pseudoviruses useful for transferring genetic material into target cells of an organism

The subject of the invention is new vectors for delivering geneticmaterial for use i.a. in gene therapy, in immunotherapy and as atherapeutic or prophylactic vaccine.

In vertebrates, the transfer of genetic material, regardless of itsultimate usefulness, may be achieved by various procedures which, forthose most widely known, are (i) transfer by viral vectors, (ii)transfer via packaging into liposomes and the like, (iii) transfermediated by facilitating agents such as cationic lipids, gold beads orcalcium phosphate and (iv) transfer by mere injection of naked DNA, thatis to say DNA lacking any other components which may interact orcooperate with the DNA in order to promote its transfer.

Each method is of general application; however, one method rather thananother may appear more appropriate depending on various factors such asthe type of material to be transferred, the site where it is desired toexpress this material, the permanent or transient nature of theexpression.

For example, if it involves correcting a genetic deficiency in anindividual, an integrative mode of transfer using viral vectors derivedfrom retroviruses may be preferred.

In other cases, for example in the treatment of cancers, a transientexpression targeted at the site of the tumour will be favoured. To thisend, viral vectors such as vaccine vectors are particularly appropriate.

In the case of vaccinal treatments, vaccine vectors, liposomes or evennaked DNA may be suitable. The latter will be preferred to retroviralvectors, in particular for preventive vaccination.

It has now been discovered that the capsids of papillomaviruses may bereconstituted in vitro, in the presence of heterologous RNA or DNA, andthat this genetic material became efficiently packaged therein. Thus,the capsids, commonly called VLPs for virus-like particles, can serve asvehicle for the transfer of genetic material, with various applications.

Papillomaviruses are nonenveloped small DNA viruses with an icosahedralstructure. Their genome codes for up to eight early proteins and twolate proteins. Their open reading frames are classified from E1 to E7without forgetting L1 and L2. The early (E for early) genes areassociated with the viral replication and cellular transformationfunctions. The papillomavirus capsids consist of two proteins L₁ and L₂(L for late proteins); L₁ being the major constituent. Detailedinformation may be found in Virology, Second Ed. by B. N. Fields, RavenPress (1990).

VLPs which mimic in every respect the capsids of native virions may beobtained by recombinant expression of either L₁ alone, or of L₁+L₂, inthe vaccine system (Hagensee et al., J. Virol. (1993) 67: 315) or in thebaculovirus system (Kirnbauer et al., PNAS (1992) 89: 12180; Kirnbaueret al., J. Virol. (1993) 67: 6929; Rose et al., J. Virol. (1993) 67:1936; Le Cann et al., FEMS Microbiol. Lett. (1994) 117: 269).

Since these VLPs adopt a native conformation and react with neutralizingantibodies known to recognize conformational epitopes present in thenative virions, it has already been suggested to use these VLPs asvaccines against papillomavirus infections (WO 94/5792).

Many animal species, including humans, are subject to papillomavirusinfections. These infectious agents are specific for the group whichthey infect. Thus, it is possible to distinguish between, inter alia,bovine papillomaviruses and human papillomaviruses (HPV). In humans,different types of HPV are responsible for various diseases. Types 1, 2,3, 4, 7, 10, and 26-29 are the cause of benign verrucas. Types 5, 8, 9,12, 14, 15, 17, 19-25, 36, and 46-50 can induce lesions inimmunologically deficient individuals. Types 6, 11, 34, 39, 41-44 and51-55 are responsible for dysplasia or nonmalignant condyloma of thegenital and respiratory mucous membranes; in rare cases, some of thesetypes may be involved in invasive carcinomas. Finally, types 16 and 18and, to a lesser extent, 31, 33, 35 and 45 cause epithelial dysplasia ofthe genital mucous membrane and are very widely associated with themajority of invasive carcinomas.

The present invention provides, for its part, noninfectiouspapillomavirus virus-like particles (VLPs) which comprise a capsiddefining an internal space and a nucleic acid molecule contained in thisinternal space; the nucleic acid molecule being different from thegenome of a papillomavirus at least in that it lacks all or part of theregions of the said genome coding for wild-type late proteins.

For the purposes of the present invention, the capsid is mainly made ofall or part of a protein L1 or of all or part of a protein L1 and all orpart of a protein L2. For the sake of simplicity, only the L1 or L2protein will be used in the text which follows to designate the wholeproteins as well as fragments thereof. It can also be expected thatthere will be several L1 or L2 proteins obtained from different types.

Among the HPV types from which the L1 and L2 proteins may be derived,there may be mentioned in particular types 1, 6, 10, 11, 16, 18, 31, 33,35 or 45.

When the proteins are obtained from an HPV-16, -18, -33 or -35 or fromany other HPV capable of inducing an invasive carcinoma, it ispreferable that the sequence of the L1 protein in use for the purposesof the invention is identical to that of the L1 protein which is presentin the papillomavirus when the latter is initially isolated from abenign lesion (e.g. condyloma acuminatum or cervical dysplasia). Indeed,it in fact appears that at the stage of a benign lesion, thepapillomavirus can still freely replicate in the complete virion state,whereas at the malignant stage, this function will be impaired in thevirus in particular because of a mutation which would have occurred inthe ORF coding for L1. This mutation would prevent, inter alia, theformation of the capsids. The sequence of a type 16 L1 protein obtainedfrom an HPV isolated from a condyloma is disclosed in the sequenceidentifier No. 2 of application WO 94/5792. It can be noted that thissequence is distinguishable from that of an L1 protein of an HPV-16isolated from a malignant carcinoma in that the amino acid at position202 is an amino acid other than histidine, i.e. an aspartic acid orglutamic acid residue.

As regards the L2 protein, the latter may be possibly deleted for itsDNA binding site in order to promote the elimination of any trace of DNAduring the purification of the components necessary for using the VLPsaccording to the invention. In practice, this involves suppressing ormodifying one or several of the first 12 amino acids of the N-terminalend. Such L2 proteins are in particular described in WO 95/20659 andZhou et al., J. Virol. (1994) 68: 619.

Alternatively, the capsid may consist of one or more hybrid proteins(fusion proteins) corresponding to the chimeras L1-E6, L1-E7, L2-E6,L2-E7 or to any other chimera form in which at least part of an L1 or L2protein may exist combined with a peptide or polypeptide heterologous toL1 or L2, for example an HIV (human immunodeficiency virus) gag peptide.In order to form such hybrids, several types of association are possiblein theory.

For example, it is possible to envisage combining, by a peptide bond,the N-terminal or C-terminal end of the whole L1 and L2 protein with theopposite end of the E6 or E7 protein. The same action can be expectedwith truncated proteins. The insertion of all or part of E6 or E7 intothe centre of the sequence of the L1 or L2 protein can also be expected,still by a peptide bond; preferably, fragments of E6 or E7 correspondingto remarkable epitopes will be inserted. The insertion into the sequenceof the L1 or L2 protein can be carried out while preserving the entireL1 or L2 sequence or alternatively by deleting a portion thereof.Obviously, the construction of appropriate expression cassette (bygenetic fusion) coding for these hybrid proteins will preside over theproduction of these proteins.

As previously mentioned, the component(s) constituting the capsid may beproduced in recombinant systems, bacteria, yeast, mammalian or insectcell. For example, WO 95/31476 deals with the expression andpurification of an L1 protein in and from E. coli. The expression andpurification in and from yeast, of the L1 proteins of HPV-6a, -11, -16and -18 is described in WO 95/31532, as well as the co-expression andthe co-purification of these same proteins with the corresponding L2proteins. The expression of the L1 protein or of the L1 and L2 proteinsof type 16, in mammalian cells, with the aid of a vaccine vector, isdescribed in WO 93/2184 and Zhou et al., Virology (1991) 185: 251. Theexpression of the type 1 L1 protein, in mammalian cells COS, with theaid of the plasmid pSVL is described in WO 94/152 and Ghim et al.,Virology (1992) 190 : 548. The expression of the type 1 L1 protein, bymeans of the vaccine system, is also disclosed by Hagensee et al., J.Virol. (1993) 67: 315. he expression of the type 16 L1 protein and itsco-expression with the corresponding L2 protein, in insect cells, withthe aid of a baculovirus, is described in WO 94/5792 and Kirnbauer etal., J. Virol. (1993) 67: 6929. On the same subject, Xi et al., J. Gen.Virol. (1991), 72: 2981, may also be mentioned. The expression of thetype 11, 16 and 18 L1 protein, in the same system, is reported by WO94/20137 and Rose et al., J. Virol. (1993) 67: 1936. Thus, thedevelopment of a recombinant system intended for the expression of an L1protein or of the L1 and L2 proteins is clearly within the capability ofpersons skilled in the art.

When these proteins are produced in a prokaryotic system, they generallyremain in the dissociated state after purification. There is noformation of VLPs unless if these proteins are subjected to a specificrenaturation treatment, and even in this particular case, the yieldremains very low.

When these proteins are expressed in a eukaryotic system, there isgenerally an expectation for the proteins produced to reassemblespontaneously in the form of VLPs, except for example if the level ofexpression was too low. Consequently, the product which is obtainedafter purification is indeed VLPs and not dissociated proteins.

In order to implement the subject of the present invention, the VLPsproduced in a eukaryotic system should therefore be treated so as todissociate them into their components. The dissociation requires thatthe disulphide bridges are reduced and that the calcium ions are removed(Volpers et al., J. Virol. (1995) 69: 3258 and Colomar et al., J. Virol.(1993) 67: 2779). For example, the VLPs will be placed at alkaline pH ora reducing agent such as dithiothreitol (DTT) will be used. Acalcium-complexing chelating agent such as EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) will also be used.

For the purposes of the present invention, the nucleic acid encapsulatedmay be RNA or DNA; the latter will be mainly preferred. The size of themolecule is not critical; it should be stated however that it ispreferable that it does not exceed 8 kbp, at least as regards the DNA.

A nucleic acid molecule which is useful for the purposes of the presentinvention should be different from the papillomavirus genome, althoughit can contain some components thereof. In particular, this moleculedoes not have the structure of a papillomavirus genome and does notcontain a replication origin specific for a papillomavirus.

The DNA may be in a linear or circular form; the latter form beingpreferred. Advantageously, it will be a plasmid. The latter will beintegrative or not, depending on the desired aim. Likewise, it may ormay not replicate in a mammalian cell. For production purposes, it willcomprise e.g. a prokaryotic replication origin.

The DNA molecule, e.g. the plasmid, may optionally comprise a site whichallows it to bind to the E2 protein of a papillomavirus. Such a site mayhave as sequence the formula ACCN₆MT in which N is independently A, G, Cor T and M is G or T. The DNA molecule may also comprise all or part ofthe long control region (LCR) of the genome of a papillomavirus.

The essential function of this DNA (or RNA) molecule is to allow theexpression of one or more peptides, polypeptides or proteins of interestin a mammalian cell. Consequently, it comprises a coding region placedunder the control of an appropriate promoter. By way of example, theremay be mentioned the human cytomegalovirus early promoter described inparticular in the American Patent U.S. Pat. No. 5,168,062 or atissue-specific promoter such as the promoter of the gene coding forhuman desmin (Li et al., Géne (1989) 78: 243 and Li et al., Development(1993) 117: 947).

The choice of the coding region will be determined by the intended useof the VLPs according to the invention. Thus, these VLPs can be used asa vaccination agent against parasitic, bacterial or viral infections. Inthis case, the peptide or polypeptide or the protein will be selectedfrom parasitic, bacterial or viral antigens.

According to a specific embodiment, the use of the VLPs according to theinvention as a therapeutic or preventive vaccination agent againstpapillomavirus infections is chosen. For this, the peptide(s),polypeptide(s) or protein(s) encoded will be advantageously selectedfrom all or part of the E1 and E2 proteins and nononcogenic forms of theE6 and E7 proteins of a papillomavirus; preferably of a type 16, 18, 31,33, 35 or 45 HPV. This papillomavirus may be optionally of a typedifferent from the one from which the capsid protein(s) is (are)derived.

The nononcogenic forms include the E6 and E7 proteins of a nononcogenicpapillomavirus as well as the deleted forms of an E6 or E7 protein of anoncogenic papillomavirus; advantageously, such a deleted form of an E6protein does not comprise all or part of the E6 region between aminoacid residues 106 and 115 (for example, it may be an HPV-16 E6 Δ(106-110) or Δ (111-115) or Δ (106-115) protein). Likewise, a deletedform of an E7 protein does not comprise all or part of the E7 regionbetween amino acid residues 20 and 26 (for example it may be an HPV-16E7 Δ (21-24) or Δ (21-26) protein).

These early proteins, their corresponding DNA fragment as well as theirnononcogenic form are described in Crook et al., Cell (1991) 67: 547 andMunger et al., EMBO J. (1998) 8: 4099.

By way of example, various possible combinations as regards the originof the proteins are presented below (nonexhaustive presentation):

Capsid Nucleic acid L1 L2 E6 E7 HPV-16 HPV-16 HPV-16 HPV-16 HPV-16HPV-16 HPV-18 HPV-18 HPV-16 and HPV-16 HPV-16 and HPV-18 and HPV-18HPV-18 HPV-18 HPV-16 and — HPV-33 — HPV-18 HPV-16 — HPV-16 — HPV-16 — —HPV-16

In another aspect, it is also possible to envisage using the VLPsaccording to the invention as a vaccination agent against tumoursinduced by autoantigens, preventively or therapeutically. Among theantigens associated with tumours, there may be mentioned in particulartyrosinase, the glycoprotein gp100, the MAGE protein family, CEA, theras protein, mutated or otherwise, the p53 protein, mutated orotherwise, Muc1 and pSA.

VLPs according to the invention may also be highly useful for deliveringin vivo cytokines or secondary molecules having an immunomodulatoryfunction (e.g. cellular recognition by the helper T cells), in allapplications where these molecules are prescribed. Among the cytokines,there may be mentioned in particular interleukin-2 (IL-2), IL-4, -5, -7,-10, -12, GM-CSF (granulocyte macrophage colony stimulating factor),gamma interferon (gamma-IFN) and TGF-beta (tumour growth factor-beta).Among the secondary molecules, there may be mentioned in particularB7.1, B7.2, CD40, CD28 and CIITA.

For example, a nucleic acid molecule useful for the purposes of thepresent invention may not only comprise a region coding for an antigenof an infectious agent or of an autoantigen associated with a tumour,but also a region coding for a cytokine, e.g. IL-2 or IL-12. In order totreat or prevent papillomavirus infections, such a region may be addedto the nucleic acid molecule as previously envisaged. In general, thismay also be carried out for any other vaccinal application.

Likewise, VLPs according to the invention whose nucleic acid moleculewould essentially code for at least one cytokine or at least onesecondary molecule can be useful in therapy as a component for treatingvarious pathologies such as tumours or autoimmune diseases oralternatively for preventing a rejection after a transplant.

Finally, VLPs according to the invention may also be useful in thetreatment of genetic diseases. In this particular case, forencapsidation, a nucleic acid molecule is prepared which comprises atleast one region coding for a protein of interest correcting a geneticdefect, such as factor VIII, for treating haemophilia, dystrophin fortreating Duchenne's muscular dystrophy (myopathy) or the protein CFTR(cystic fibrosis transmembrane regulator) for treating cystic fibrosis.

Consequently, the subject of the invention is also:

(i) as a medicament, a VLP according to the invention;

(ii) a pharmaceutical composition comprising, as active ingredient, atleast one VLP according to the invention in combination with apharmaceutically acceptable diluent or carrier;

(iii) a pharmaceutical composition comprising at least two VLPs, inwhich a first VLP comprises a capsid consisting of at least all or partof the L1 protein of a first type, such as type 16 and in which a secondVLP comprises a capsid consisting of at least all or part of the L1protein of a second type different from the first type, such as type 18;

(iv) the use of a VLP according to the invention, in the preparation ofa medicament for the prevention or treatment of a bacterial or viralinfection, of a tumour i.a. induced by an autoantigen or of anautoimmune disease or alternatively, for the prevention of graftrejection;

(v) a method for the treatment or prevention of a bacterial or viralinfection, of a tumour i.a. induced by an autoantigen or of anautoimmune disease or alternatively, for the prevention of graftrejection, according to which a therapeutically or prophylacticallyeffective quantity of at least one VLP according to the invention isadministered to an individual needing such a treatment; and

(vi) a method of in vivo expression, which makes it possible to providea mammal with a peptide, a polypeptide or a protein in a physiologicallyactive form, according to which at least one VLP according to theinvention in which the nucleic acid molecule comprises a region codingfor the said peptide or polypeptide or for the said protein, placedunder the control of an appropriate promoter if a DNA molecule isinvolved, is administered to the mammal.

A composition according to the invention may be manufactured in aconventional manner. In particular, at least one VLP is combined with apharmaceutically acceptable diluent or carrier. Examples of diluents orcarriers as well as of methods of formulation are indicated inRemington's Pharmaceutical Sciences. The formulation may depend on theroute of administration; aerosol, injectable formulation, suppositories,tablets and the like.

A composition according to the invention may be administered by anyconventional route in use in the field of vaccines, when thiscomposition is intended to this effect. They are in particular thesystemic routes, e.g. subcutaneous, intradermal, intramuscular orintravenous route, and mucosal routes, e.g. oral, nasal, pulmonary oranogenital route. When the treatment of solid tumours is involved, theabovementioned routes continue to be used and the intratumour route mayalso be added thereto. When the treatment of genetic diseases isinvolved, the choice of the route of administration will essentiallydepend on the nature of the disease; for example, there may beadvantageously mentioned the pulmonary route in the case of cysticfibrosis (the VLPs being formulated in aerosol form) or the intravenousroute in the case of haemophilia.

The administration may be carried out at a single dose or at a doserepeated once or several times after a certain time interval. Theappropriate dosage varies according to various parameters, for examplethe individual treated or the mode of administration. In general, onedose comprises from 1 to 250 μg of VLPs according to the invention.

The invention also relates to a method of preparing VLPs according tothe invention, according to which a nucleic acid molecule as definedabove is mixed with all or part of the L1 protein of a papillomavirus indissociated form and, optionally, all or part of the L2 protein of apapillomavirus, in the presence of an agent allowing the reassociationof the L1 protein (or of the L1 and L2 proteins) in capsid form, e.g. acalcium salt, and the said VLPs are recovered from the mixture.

When the DNA intended to be encapsidated comprises a site which allowsit to bind to the E2 protein, it becomes advantageous to add thisprotein to the reconstitution mixture. This protein would, for example,have been previously produced by the recombinant route, in a prokaryotic(bacteria) or eukaryotic (i.a. yeast, insect cells) system.

Prior to the mixing step, it is advantageous to express all or part ofthe L1 protein, optionally all or part of the L2 protein, by therecombinant route in a eukaryotic host cell. In this case, the emptyvirus-like particles are recovered and they are treated with a reducingagent and/or with a calcium-ion chelating agent in order to obtain allor part of the L1 protein, optionally all or part of the L2 protein, indissociated form.

Advantageously, all or part of the L₁ protein, optionally all or part ofthe L₂ protein, is expressed by the recombinant route in insect cellsinfected with a baculovirus into whose genome a DNA fragment is insertedwhich codes for all or part of the L1 protein, optionally for all orpart of the L2 protein, placed under the control of an appropriatepromoter.

When the VLPs according to the invention are used in long-termtreatments, such as for example in the treatment of a cancer, therepeated administration of the same type of VLPs (that is to say of VLPshaving the same capsid) may be problematic from an immunological pointof view. In order to overcome this potential disadvantage, it ispossible to envisage the sequential use of VLPs having differentcapsids. For example, it is possible to prepare a whole range of VLPshaving the same nucleic acid molecule (having a region coding e.g. forIL-2 or IL-12) but differing in the type of papillomavirus from whichthe L1 protein and optionally the E2 protein are derived. Thus, therewill be used in succession type 16 capsid VLPs (once or several times),and then type 18 capsid VLPs (once or several times) and the like.

Accordingly, the subject of the invention is also:

(i) a method of treating a genetic disease, a cancerous state or apapillomavirus infection according to which the VLPs according to theinvention are repeatedly administered to a mammal needing such atreatment at t_(n), t_(n+1); n being a number greater than or equal to1; the VLPs administered at t_(n+1) differing from the VLPs administeredat t_(n) in that the L₁ or the L₁ and L₂ proteins of the capsid of theVLPs administered at t_(n+1), is (are) derived from a papillomavirus ofa type other than that from which the L₁ protein or the L₁ and L₂proteins of the capsid of the VLPs administered at t_(n) is (are)derived; and

(ii) a pharmaceutical composition which comprises several products foradministration in succession; the products each consisting of VLPsaccording to the invention and differing from each other in that the L₁protein or the L₁ and L₂ proteins of the capsid of the VLPs is (are)derived for each product from a different type of papillomavirus.

EXAMPLE Preparation of Empty VLPs

A stock of HPV-16 type VLPs is prepared from a culture of Sf-9 cellsinfected with a recombinant papillomavirus. This baculovirus possesses,inserted into its genome, the DNA fragments of HPV-16 coding for L₁ andL₂, which were originally isolated from a condylomata acuminata. Thesequence coding for L₁ is disclosed in WO 94/5792. It should be noted inparticular that the codon corresponding to the amino acid at position202 is an aspartic acid codon.

The construction of the baculovirus, the culture of the Sf-9 cells aswell as the purification of the VLPs are described in Kirnbauer et al.,J. Virol. (1993) 67: 6929 or in Suzich et al., PNAS (1995) 92: 11553.

Dissociation of the VLPs

250 μl of 1 mM phosphate buffer pH 8 containing 300 mM NaCl, 2 mM EGTA(ethylene glycol tetraacetic acid) and 40 mM DTT (dithiotreitol) areadded to 250 μl of a preparation of VLPs obtained after dialysis againsta 1 mM phosphate buffer pH 8. The incubation is left to continue at 37°C. for one hour. The final concentration of VLPs subjected todissociation is of the order of 200 μg/ml. A variant of the dissociationprotocol is also described in Volpers et al., J. Virol. (1995) 69: 3258.This preparation is then dialysed extensively against 1 mM phosphatebuffer pH 8.

Preparation of the DNA Intended to be Encapsidated

The plasmid pnRSV-NP (A/PR/8/34) which comprises the cDNA coding for thenucleoprotein of the influenza virus A/PR/8/34 under the control of theRous sarcoma virus (RSV) promoter is prepared as described in Ulmer etal., Science (1993) 259: 1745.

Encapsidation of the DNA

50 ng of purified DNA in a volume of 500 μl are added to 500 μl of thepreparation of dissociated VLPs obtained above. Next, 25 μl of a 20 mMcalcium chloride solution are added. The mixture is incubated for 30 minat 37° C. It is then subjected to centrifugation in 40% sucrose in anSW28 rotor at 28,000 rpm for 20 hours. A band is recovered at thedensity of 1.33 g/ml which contains the encapsidated DNA and which isdialysed against 1 mM phosphate buffer pH 8.

What is claimed is:
 1. A noninfectious virus-like particle (VLP) whichcomprises: (i) a capsid defining an internal space and consisting of atleast a portion of the L₁ protein of a papillomavirus that has theability to self-assemble into a VLP, and (ii) a nucleic acid moleculecontained in the said internal space; the nucleic acid molecule beingdifferent from the genome of a papillomavirus at least in that it lacksall or part of the regions of the said genome coding for wild-type lateproteins.
 2. The virus-like particle according to claim 1, in which thecapsid consists of said at least a portion of the L₁ protein in theL₁-E₇ chimeric protein state.
 3. The virus-like particle according toclaim 1, in which the capsid consists of said at least a portion of theL₁ protein of a human papillomavirus (HPV).
 4. The virus-like particleaccording to claim 3, in which the capsid consists of said at least aportion of the L₁ protein of a type 1, 6, 10, 11, 16, 18, 31, 33, 35 or45 human papillomavirus.
 5. The virus-like particle according to claim4, in which the capsid consists of said at least a portion of the L₁protein of an HPV-16, -18, -31, -33, -35 or -45 initially isolated froma benign lesion.
 6. The virus-like particle according to claim 5, inwhich the capsid consists of said at least a portion of the L₁ proteinof an HPV-16 having an amino acid sequence which comprises at position202 an amino acid other than histidine.
 7. The virus-like particleaccording to claim 6, in which the capsid consists of said at least aportion of the L₁ protein of an HPV-16 having an amino acid sequencewhich comprises at position 202 an aspartic acid or glutamic acidresidue.
 8. The virus-like particle according to claim 5, wherein saidbenign lesion is condyloma acuminatum or cervical dysplasia.
 9. Thevirus-like particle according to claim 1, in which the capsid, inaddition, consists of at least a portion of the L₂ protein of apapillomavirus that has the ability to self assemble into a VLP.
 10. Thevirus-like particle according to claim 9, in which the capsid inaddition, consists of said at least a portion of the L₂ protein in theL₂-E7 chimera state.
 11. The virus-like particle according to claim 1,in which the nucleic acid molecule comprises a region coding for aprotein of interest.
 12. The virus-like particle according to claim 11,in which the nucleic acid molecule is DNA and comprises a region codingfor a protein of interest placed under the control of a promoter whichpromotes transcription in mammalian cells.
 13. The virus-like particleaccording to claim 12, in which the DNA molecule comprises, in addition,a papillomavirus E₂ protein binding site of formula ACCN₆MT in which Nis A, G, C or T and M is G or T.
 14. The virus-like particle accordingto claim 12, in which the DNA molecule comprises, in addition, all orpart of the long control region (LCR) of the genome of a papillomavirus.15. The virus-like particle according to claim 11, in which the nucleicacid molecule is at most 8 kbp.
 16. The virus-like particle according toclaim 11, in which the nucleic acid molecule comprises at least oneregion coding for a protein of interest selected from cytokines andsecondary molecules facilitating cellular recognition by the helper Tcells.
 17. The virus-like particle according to claim 11, in which thenucleic acid molecule comprises at least one region coding for a proteinof interest selected from tumour-associated antigens.
 18. The virus-likeparticle according to claim 11, in which the nucleic acid moleculecomprises at least one region coding for a protein of interest selectedfrom parasitic, bacterial or viral antigens.
 19. The virus-like particleaccording to claim 18, in which the nucleic acid molecule comprises atleast one region coding for a protein of interest selected from the E₁and E₂ proteins and the nononcogenic forms of the E₆ and E₇ proteins ofa papillomavirus.
 20. The virus-like particle according to claim 19, inwhich the nucleic acid molecule comprises at least one region coding fora protein of interest selected from the E₁ and E₂ proteins and thenononcogenic forms of the E₆ and E₇ proteins of a papillomavirus of theHPV-16, -18, -31, -33, -35 or -45 type.
 21. The virus-like particleaccording to claim 19 or 20, in which the nucleic acid moleculecomprises at least one region coding for a protein of interest selectedfrom the E₁ and E₂ proteins and the nononcogenic forms of the E₆ and E₇proteins of a papillomavirus of a type different from the one from whichthe capsid protein or protines are derived.
 22. The virus-like particleaccording claim 10, in which the nucleic acid molecule comprises atleast one region coding for a protein of interest in the therapy ofgenetic diseases.
 23. A virus-like particle as a medicament according toclaim
 1. 24. A method of preparing virus-like particles according toclaim 1, wherein a nucleic acid molecule different from the genome of apapillomavirus at least in that it lacks all or part of the regions ofthe said genome coding for the wild-type late proteins is mixed withsaid at least a portion of the L₁ protein of a papillomavirus indissociated form, in the presence of a calcium salt and the saidvirus-like particles are recovered from the mixture.
 25. The method ofpreparation according to claim 24, wherein all or part of the E₂ proteinof a papillomavirus is mixed with the nucleic acid molecule and with theL₁ protein.
 26. The method of preparation according to claim 25, whereinthe papillomavirus E₂ protein deleted for its N-terminal part is addedat the time of mixing.
 27. The method of preparation according to claim24, wherein prior to the mixing step, (i) said at least a portion of theL₁ protein is expressed by the recombinant routes (ii) the emptyvirus-like particles are recovered and (iii) the empty virus-likeparticles are treated with a reducing agent and/or with a calcium-ionchelating agent in order to obtain said L₁ protein in dissociated form.28. The method of preparation according to claim 27, in which said atleast a portion of the L₁ protein is expressed by the recombinant routein insect cells infected with a baculovirus into whose genome a DNAfragment is inserted which codes for said L₁ protein placed under thecontrol of an appropriate promoter.
 29. The method of preparationaccording to claim 28, wherein said preparation further comprises atleast a portion of the L₂ protein that has the ability to self assembleinto a VLP to be expressed by the recombinant route in insect cellsinfected with a baculovirus into whose genome a DNA fragment is insertedwhich codes for said L₂ protein placed under the control of anappropriate promoter.
 30. The method of preparation according to claim27, wherein step (i) further comprises at least a portion of the L₂protein that has the ability to self assemble into a VLP to be expressedby the recombinant route and wherein step (iii) further comprises saidL₂ protein in dissociated form.